Hans Aerts

Plasma level of the macrophage-derived soluble CD163 is increased and positively correlates with severity in Gaucher's disease

Recently, soluble CD163 (sCD163) has been identified as a macrophage/monocyte‐specific plasma protein and increased concentrations have been measured in patients with infection and myeloid leukaemia. In the present study we investigated the levels of sCD163 in patients with Gauchers disease, an inherited lysosomal storage disorder characterised by hepato‐ and spleno‐megaly due to excessive accumulation of macrophages. The sCD163 plasma levels, median (25–75 percentiles), were far above the levels in normal subjects [7.1 mg/L (4.8–10.3) vs. 1.9 mg/L (1.5–2.4), P < 0.0001]. After initiation of enzyme supplementation therapy, the sCD163 levels were significantly reduced [4.7 mg/L (3.2–6.6), P = 0.0004]. sCD163 correlated with disease severity (rho = 0.43, P < 0.0061) and chitotriosidase activity (rho = 0.71, P > 0.0001). This study further establishes that sCD163 may be a valuable laboratory parameter in monitoring disease with increased macrophage activity.

Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

Expression of the pfmdr1-encoded Pgh1 protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pgh1-mediated accumulation of this antimalarial. Pgh1 carrying amino acid substitutions associated with chloroquine resistance in P. falciparum does not confer this phenotype. Here, we present studies on the underlying mechanism of Pgh1 mediated chloroquine influx into CHO cells. First, we measured intralysosomal pH using FITC-labelled dextran and found the intralysosomal pH in Pgh1 expressing cells to be decreased. A decreased lysosomal pH was not observed in cells expressing Pgh1 carrying the S1034C and N1042D double substitution found in some chloroquine-resistant P. falciparum parasites. Secondly, Pgh1-mediated uptake of chloroquine was abolished in the presence of bafilomycin A1, a specific inhibitor of vacuolar [H+]ATPases and was nearly abrogated in the presence of NH4Cl. Finally, cells expressing wild-type Pgh1 showed increased uptake of both (+)- and (-)[3H]chloroquine enantiomers, indicating that Pgh1-mediated uptake of chloroquine is not enantioselective and in agreement with a pH-driven process. We conclude from these studies that Pgh1 does not transport chloroquine, but instead influences chloroquine accumulation by modulating the pH of acidic organelles. This function is abolished in Pgh1 carrying amino acid substitutions S1034C and N1042D. We speculate that the pfmdr1 gene encodes a vacuolar chloride channel.

Subcellular fractionation of cultured normal human melanocytes: New insights into the relationship of melanosomes with lysosomes and peroxisomes

In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems, i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.

Dyscholesterolemia Protects Against Ischemia-Induced Ventricular Arrhythmias

Background—Hypercholesterolemia protects against ventricular fibrillation in patients with myocardial infarction. We hypothesize that hypercholesterolemia protects against ischemia-induced reentrant arrhythmias because of altered ion channel function. Methods and Results—ECGs were measured in low-density lipoprotein receptor knockout (LDLr−/−), apolipoprotein A1 knockout (ApoA1−/−), and wild-type (WT) mice. Action potentials, calcium handling, and ion currents were recorded in ventricular myocytes. Gene expression was determined by quantitative polymerase chain reaction and Western blot. In isolated perfused hearts, regional ischemia was induced and arrhythmia inducibility was tested. Serum low-density lipoprotein (LDL) cholesterol was higher in LDLr−/− mice than in WT mice (2.6 versus 0.4 mmol/L), and high-density lipoprotein cholesterol was significantly lower in ApoA1−/− mice than in WT mice (0.3 versus 1.8 mmol/L). LDLr−/− and ApoA1−/− myocytes contained more cholesterol than WT (34.4±2.8 and 36.5±2.4 versus 25.5±0.4 &mgr;mol/g protein). The major potassium currents were not different in LDLr−/− and ApoA1−/− compared with WT mice. The L-type calcium current (ICa), however, was larger in LDLr−/− and ApoA1−/− than in WT (12.1±0.7 and 12.8±0.8 versus 9.4±1.1 pA/pF). Calcium transient amplitude and fractional sarcoplasmic reticulum calcium release were larger and action potential and QTc duration longer in LDLr−/− and ApoA1−/− than in WT mice (action potential duration at 90% of repolarization: 102±4 and 106±3 versus 84±3.1 ms; QTc: 50.9±1.3 and 52.8±0.8 versus 43.5±1.2 ms). During ischemia, ventricular tachycardia/ventricular fibrillation inducibility was larger in WT than in LDLr−/− and ApoA1−/− hearts. Expression of sodium channel and Ca-handling genes were not significantly different between groups. Conclusions—Dyscholesterolemia is associated with action potential prolongation because of increased ICa and reduces occurrence of reentrant arrhythmias during ischemia.

Abstract 4800: Quantitative detection of discriminatory proteins in tissue and serum of ovarian cancer patients by LC-MSE

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Using label-free UPLC-MSE quantification methods to identifying proteins as well as determining their abundance in serum and microdissected ovarian cancer tissue. Experimental procedures: Serum of six patients with a serous adenocarcinoma of the ovary (stage IIIB or higher) was collected before treatment. Matched control patients with a serous cystadenoma of the ovary were chosen as a control group. In addition, homogeneous regions of cells that exhibited uniform histology were isolated from cancer tissue by laser capture microdissection (LCM), normal ovarian epithelial cell samples and stroma samples were taken from matched controls. We subsequently employed label-free ultra-high pressure liquid chromatography tandem mass spectrometry (UPLC-MSE) to identify proteins and determine their absolute concentrations in serum and tissue lysates. Moderated t-tests were used to identify differentially expressed proteins between patient- and control-group. p-values were adjusted for multiple-testing using Benjamini-Hochberg false discovery rates and considered differentially expressed when <0.05. Summary of the data: In the serum specimens, 13 differentially expressed proteins were identified by LC-MSE profiling. Proteins with different concentrations in patients versus control sera include abundant serum proteins such as apolipoprotein AI and transferrin, both exhibited a lower concentration in serum of cancer patients. Differential expression was also observed for apolipoproteins (APOA IV, APOA II and APOC III) and other proteins that have not been associated with ovarian cancer previously such as C9 and Afamin. In the tissue-lysates we identified 535 Proteins of which 332 could be reliably quantified. Of these 56 were differentially expressed proteins of which 12 had non-overlapping standard deviations. Literature searches confirmed a link between ovarian cancer and 6 of these proteins, suggesting a possible role in disease progression. Of special interest was the identification of prohibitin (PHB) and cofilin-1 (CFL1). Immunohistochemical detection of CFL1 in formalin-fixed paraffin-embedded tissues of our patient group showed heterogeneous expression in 50% of the serous ovarian carcinomas, whereas all the benign lesions were negative. These results again demonstrate the validity of the protein quantitation results obtained by MSE. Interestingly, no overlap was found between the discriminatory proteins in serum specimens and corresponding tissue lysates, illustrating the challenge to identify a single biomarker in the circulation reflecting ovary cancer. Conclusions: Our study revealed several protein changes in serum and tissue of patients suffering from serous adenocarcinoma of the ovary. Further investigation of these proteins is warranted to establish whether they could provide new insights into disease etiology and act as potential new markers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4800. doi:1538-7445.AM2012-4800

Mannose Phosphate-Independent Membrane Association of Lysosomal Enzymes Occurs After Passage of the Golgi Complex

The degradative action of lysosomes depends primarily on the hydrolytic enzymes present within their lumen. However, the number of lysosomes per cell and their level of activity is regulated by events controlled by constituents of their limiting membrane. Therefore, study of formation of new lysosomes, the composition of lysosomal membranes and their maintenance is the basis for understanding the biology of lysosomes. Much is known about the biosynthesis, sorting and turnover of soluble lysosomal enzymes (Von Figura and Hasilik, 1986; Kornfeld and Mellman, 1989). They are targeted through endosomes to lysosomes via the mannose phosphate receptor (MPR). In the endosomes MPR separates from the ligand (the soluble lysosomal enzyme) and returns to the Golgi complex or the cell surface, while the enzyme is transported to the lysosomes. Other mechanisms to direct proteins to the lysosomes must exist (Neufeld and McKusik, 1983). The glycoproteins of the limiting lysosomal membrane have no mannose 6-phosphate and yet are very efficiently transported to the lysosomes. In addition, some enzymes such as glucocerebrosidase (deficient in Gaucher disease) and acid phosphatase have their own unique pathways to reach the lysosomes (Erickson, et al., 1985; Braun et al., 1989). We We have studied the intracellular transport between the Golgi complex and the lysosomes of two lysosomal glycoproteins cathepsin D, a MPR-dependent soluble enzyme and glucocerebrosidase, a MPR-independent enzyme after their synthesis in the rough endoplasmic reticulum (RER).